Tropical zooplankton in the highly-enclosed lagoon of Taiaro Atoll (Tuamotu Archipelago, French Polynesia)

 Nocturnal zooplankton assemblages around Taiaro Atoll were sampled over six nights during February 1994. Replicate zooplankton samples were collected at windward and leeward locations in the enclosed lagoon and adjacent ocean with a metered net (85 cm diameter, 500 μm mesh) towed for 15 min at 5 m depth. The zooplankton community in the lagoon was very different from that in the ocean. Oceanic samples contained 50 mostly holoplanktonic taxa (diversity index, H′=2.62; evenness index, J′=0.67). Lagoonal samples contained 19 mostly meroplanktonic taxa (H′=1.54, J′=0.52) with three taxa (decapod larvae; an ostracod, Cypridina sp.; a copepod, Acartia fossae) contributing >90% of the individuals. Unlike the ocean, zooplankton distributions in the lagoon were not homogenous; instead spatial patterns were apparently formed by the interaction between hydrodynamic processes and species-specific behaviour. Accepted: 28 August 1997     

Distribution of ten antibiotic resistance genes in <Emphasis Type="Italic">E. coli</Emphasis> isolates from swine manure,lagoon effluent and soil collected from a lagoon waste application field

The prevalence of ten antibiotic resistance genes (ARGs) was evaluated in a total of 616 Escherichia coli isolates from swine manure, swine lagoon effluent, and from soils that received lagoon effluent on a commercial swine farm site in Sampson County, North Carolina (USA). Isolates with ARGs coding for streptomycin/spectinomycin (aadA/strA and strB), tetracycline (tetA and tetB), and sulfonamide (sul1) occurred most frequently (60.6–91.3%). The occurrence of E. coli isolates that carried aadA, tetA, tetB, and tetC genes was significantly more frequent in soil samples (34.0–97.2%) than in isolates from lagoon samples (20.9–90.6%). Furthermore, the frequency of isolates that contain genes coding for aadA and tetB was significantly greater in soil samples (82.6–97.2%) when compared to swine manure (16.8–86.1%). Isolates from the lagoon that carried tetA, tetC, and sul3 genes were significantly more prevalent during spring (63.3–96.7%) than during winter (13.1–67.8%). The prevalence of isolates from the lagoon that possessed the strA, strB, and sul1 resistance genes was significantly more frequent during the summer (90.0–100%) than during spring (66.6–80.0%). The data suggest that conditions in the lagoon, soil, and manure may have an impact on the occurrence of E. coli isolates with specific ARGs. Seasonal variables seem to impact the recovery isolates with ARGs; however, ARG distribution may be associated with mobile genetic elements or a reflection of the initial numbers of resistant isolates shed by the animals.     

Biodegradation of dichloromethane by the polyvinyl alcohol-immobilized methylotrophic bacterium Ralstonia metallidurans PD11

A dichloromethane (DCM)-degrading bacterium, Ralstonia metallidurans PD11 NBRC 101272, was immobilized in a polyvinyl alcohol (PVA) gel to use in a bioreactor for DCM treatment. After 4-month incubation of PVA gel beads with R. metallidurans PD11 and DCM in a mineral salt medium, the cells were tightly packed in the mesh of the gel. Forty beads of the gel in 10 ml of a batch system model showed effective activity degrading 500 and 1,000 mg l⁻¹ DCM within 2 and 3 h, respectively. Although reduction of pH due to accumulation of chloride ion liberated from DCM decreased the activity, it was recovered by adjustment to neutral pH. The activity of the immobilized cells was not affected by addition of nutrients which were preferentially utilized by R. metallidurans PD11, unlike the activity of the free-living cells. A continuous flow system with a column was more effective for rapid degradation of DCM. Thus, the PVA gel–immobilized cell of R. metallidurans PD11 is thought to be a prospective candidate to develop the bioreactor.     

Antibiotic resistance and molecular characterization of clinical isolates of methicillin-resistant coagulase-negative staphylococci isolated from bacteremic patients in oncohematology

Polymerase chain reaction (PCR) amplification of antibiotic resistance genes as well as staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA were used to characterize 45 methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates responsible of bacteremia recovered in patients at the Bone Marrow Transplant Centre of Tunisia in 1998–2007. Among the 45 MRCoNS isolates, Staphylococcus epidermidis was the most prevalent species (75.6%) followed by Staphylococcus haemolyticus (22.2%) and Staphylococcus hominis (2.2%). Extended susceptibility profiles were generated for MRCoNS against 16 antimicrobial agents. Out of 45 mecA-positive strains, 43 (95.6%) were phenotypically methicillin-resistant and two (4.4%) were methicillin-susceptible. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm(A) and erm(B) in two (7.4%) isolates. The aac(6′)-Ie-aph(2″)-Ia was the most prevalent gene among aminoglycoside-resistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4′)-Ia in 18 (42.8%) isolates, in combination with aph(3′)-IIIa in four (9.5%) or with both ant(4′)-Ia and aph(3′)-IIIa in two (4.7%) isolates. The ant(4′)-Ia was detected in three (7.1%) isolates and the aph(3′)-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet(M) genes. SCCmec types IV (31%) and III (24.5%), the most prevalent types detected, were found to be more resistant to non-β-lactam antibiotics. A wide diversity of isolates was observed by PFGE among MRCoNS.     

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis> Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.     

Microorganisms in a high altitude Glacier Ice in Tibet

Eighty-one strains of viable microorganisms were recovered from 23 samples collected from Ice Core 3 of Malan Glacier (China, 91° 45.3′ E, 35° 48.4′ N) drilled at high altitude (5620 m). All the strains were prokaryotes—75 of bacteria (including spore-forming ones) and 6 of actinomycetes. The characteristic genera differ from those of Arctic and Antarctic ice, in which many fungi and algae are widely distributed; this shows an difference of environmental conditions between Tibet and polar regions. The variation in number and species ofBacillus in different ice core layers implied changes of environmental conditions in the past.     

5′ Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae

We describe a 5′ untranslated region (5′UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5′UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5′UTRs at different temperatures. We found that the 5′UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30°C). Moreover, at high temperature (37°C), the translation efficiency of the mRNA containing the Hsp12 5′UTR was far superior to that of mRNAs containing nonheat-shock 5′UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5′UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae.     

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.     

Functional analysis of <Emphasis Type="Italic">Antirrhinum kelloggii</Emphasis> flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes; critical role in flower color and evolution in the genus <Emphasis Type="Italic">Antirrhinum</Emphasis>

The enzymes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3′5′H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3′H and two F3′5′H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3′Hs and F3′5′Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3′H and F3′5′H, respectively, and that the ancestor of A. majus lost F3′5′H activity after its speciation from the ancestor of A. kelloggii.     

Characterization of a complex restriction-modification system detected in<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains isolated from infections of domestic animals

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates ofStaphylococcus aureus andStreptococcus agalactiae obtained from clinical material. Among the 100 isolates ofS. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I andSau33 I); the targeting sequence was determined as 5′-GGN CC-3′ (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I,Sau93 I,Sau96* I,Sau98 I) and enzymes recognized sequence 5′-CTY RAG-3′ (SmlI isoschizomer). Analysis of 40 isolates ofS. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23;Sag16 I andSag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5′-CTG CA/G-3′ (PstI isoschizomer). In RMS-positiveS. aureus andS. agalactiae isolates plasmid DNA capable of replication inEscherichia coli andBacillus subtilis was also detected and isolated. This research was supported by VEGA grant of theSlovak Academy of Sciences no. 2/2059/22 and grant no. 2003 SP27/0208E 02/028/0E02 of theMinistry of Agriculture of the Slovak Republic.     

A survey of ammonia-assimilating micro-organisms in cattle manure composting

AIMS: To evaluate the ammonia-assimilating abilities of micro-organisms isolated from cattle manure composting processes and to determine the distribution of cultivable species of ammonia-assimilating micro-organisms in microbial communities during the composting processes. METHODS AND RESULTS: Compost samples were collected from four stages of treatment. Trypto soya agar was used for the isolation of ammonia-assimilating aerobes. Many of the isolates showed high ammonia-assimilating ability in a medium containing basal components and a compost extract. Partial 16S ribosomal DNA sequencing showed that the cultivable species of highly efficient ammonia-assimilating isolates changed during the composting process. The community structure of micro-organisms and actinomycetes was analysed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Two species of actinomycetes identified by PCR-DGGE coincided with those found among the cultured isolates. CONCLUSIONS: Ammonia-assimilating micro-organisms obtained by the cultivation method were not predominant in the microbial community during the composting process: however certain cultured actinomycetes were members of predominant species in the actinomycetes community. SIGNIFICANCE AND IMPACT OF THE STUDY: Ammonia assimilation by micro-organisms is one of the important mechanisms for ammonia retention in the composting process. Cultivable actinomycetes are a means for preventing ammonia emission from the composting process.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

High genetic diversity in the coat protein and 3′ untranslated regions among geographical isolates of<Emphasis Type="Italic">Cardamom mosaic virus</Emphasis> from south India

A survey was conducted to study the biological and genetic diversity ofCardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3′ terminal region consisting of the coat protein (CP) coding sequence and 3′ untranslated region (3′UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3′UTR, making CdMV one of the highly variable members ofPotyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.     

Scaling patterns of plankton diversity: a study of ciliates in a tropical coastal lagoon

Although spatial and temporal variation in plankton diversity is regularly investigated in surveys, experiments, and models, there is a lack of methods for predicting spatial patchiness of plankton diversity. We develop and apply a suite of geostatistical and multiple-regression analysis tools to assess ciliate diversity in a tropical coastal lagoon; these methods can predict spatial and temporal patterns of diversity, provide error estimates associated with these predictions, and assess which environmental factors may drive diversity patchiness. Geostatistical analysis was applied to H′ (Shannon diversity index) from 25 to 35 data collected from a sampling grid (40 × 40 m), and 10 dispersed lagoonal sites, in the dry and rainy seasons, on numerous occasions. Conditional simulation and kriging were used to predict diversity at the lagoonal and small scales, respectively. The relationship between diversity (H′) and the environment was examined by multiple-regression analysis. Thirty-six ciliate morphospecies occurred; H′ ranged from 0 to 1.9 and was patchy. Multiple regression indicated ciliate diversity changed seasonally, increasing when the sand bar was open and the lagoon was connected with the sea. Geostatistical analysis extended the recognition that seasonal changes alter diversity: when the rainy season produced a variable environment, relatively small scale patches and diversity gradients occurred; in the dry season, when the lagoon was physically uniform, larger and fewer diversity patches occurred; at the sublagoonal scale, diversity patches were similarly structured in the two seasons. Results indicate environmental variability and immigration can be the main drivers behind ciliate diversity in the lagoon. We recommend the patterns these data reveal and methods we employ be considered when further studies are continued to examine diversity on local and larger scales. Handling editor: S. Declerck     

Removal of toxic chromate using free and immobilized Cr(VI)-reducing bacterial cells of Intrasporangium sp. Q5-1

Chromate-reducing microorganisms with the ability of reducing toxic chromate [Cr(VI)] into insoluble trivalent chromium [Cr(III)] are very useful in treatment of Cr(VI)-contaminated water. In this study, a novel chromate-reducing bacterium was isolated from Mn/Cr-contaminated soil. Based on morphological, physiological/biochemical characteristics and 16S rRNA gene sequence analyses, this strain was identified as Intrasporangium sp. strain Q5-1. This bacterium has high Cr(VI) resistance with a MIC of 17 mmol l⁻¹ and is able to reduce Cr(VI) aerobically. The best condition of Cr(VI) reduction for Q5-1 is pH 8.0 at 37°C. Strain Q5-1 is also able to reduce Cr(VI) in resting (non-growth) conditions using a variety of carbon sources as well as in the absence of a carbon source. Acetate (1 mmol l⁻¹) is the most efficient carbon source for stimulating Cr(VI) reduction. In order to apply strain Q5-1 to remove Cr(VI) from wastewater, the bacterial cells were immobilized with different matrices. Q5-1 cells embedded with compounding beads containing 4% PVA, 3% sodium alginate, 1.5% active carbon and 3% diatomite showed a similar Cr(VI) reduction rates to that of free cells. In addition, the immobilized Q5-1 cells have the advantages over free cells in being more stable, easier to re-use and minimal clogging in continuous systems. This study provides potential applications of a novel immobilized chromate-reducing bacterium for Cr(VI) bioremediation.     

Characterization of an Extracellular Serine Protease Gene from the Nematophagous Fungus <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>

The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.     

Objectives and background to the 1994 Franco-Australian expedition to Taiaro Atoll (Tuamotu Archipelago, French Polynesia)

 The 9 km² uplifted lagoon of Taiaro Atoll (15°45′S, 144°38′W) is hypersaline due to its isolation from the ocean, yet it contains a high diversity of fish. The question unifying our expedition was to discover whether these assemblages could be self-sustaining despite very limited contact with the ocean. Although we were constrained by time, collections of fish larvae showed that some species can complete their life-cycle within the lagoon, while others differed genetically between the lagoon and the ocean, consistent with restricted gene flow. The lagoon contained few oceanic species of zooplankton, confirming its general isolation, but nevertheless some fish species may depend upon infrequent colonisation from the ocean (when large waves drive water over the normally dry reef crest). Isotopic signatures in fish otoliths suggest the basis for a more definitive and inclusive test of the sources of the lagoonal assemblage. Accepted: 28 August 1997     

Isolation of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) producer from Malaysian environment using γ-butyrolactone as carbon source

Samples from various natural environments in Peninsular Malaysia were screened for microorganisms that are capable of producing poly(3-hydroxybutyrate-co-4-hydroxybutyrate). A total of 663 isolates were isolated and 119 out of these isolates were identified as possible PHA producers based on Nile red staining methods. All these potential producers emitted pink fluorescence when grown on solid mineral salts medium (MSM) containing Nile red and exposed to UV light. The isolates obtained in this study were cultivated in MSM containing γ-butyrolactone as the carbon source. Gas chromatography (GC) analysis confirmed that 95 out of the 119 isolates were PHA producers. Among the 95 positive isolates, 77 isolates produced only P(3HB) homopolymer and 18 isolates produced PHA containing 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) monomers. Of these 18 isolates, USMAA1020 was screened as the best P(3HB-co-4HB) producer based on GC analysis. For further confirmation, PHA was extracted from the isolate and analyzed by GC as well as nuclear magnetic resonance (NMR). Results from both analyses confirmed that this isolate was capable of producing PHA containing 3HB and 4HB. Based on, biochemical characterization, 16S rRNA sequencing, DNA base composition, cellular fatty acids analysis and DNA–DNA hybridization, it is clearly indicated that this isolate belongs to the genus Cupriavidus. Poly(3HB-co-4HB) was synthesized by this bacterium in one-stage, two-stage and three-stage cultivation using γ-butyrolactone as the carbon source. The highest 4HB composition of 82 mol% was obtained through three-stage cultivation.